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CN-122004132-A - Method for establishing efficient regeneration system of fenugreek

CN122004132ACN 122004132 ACN122004132 ACN 122004132ACN-122004132-A

Abstract

The invention discloses a method for establishing a high-efficiency regeneration system of fenugreek, and belongs to the technical field of medicinal plant tissue culture. The method comprises the steps of (1) inoculating a fenugreek explant into a primary induction culture medium containing 5-azacytidine, spermidine and fluopicolide for induction culture to generate adventitious buds, (2) cutting the adventitious buds, transferring the adventitious buds into a proliferation culture medium containing trehalose, paclobutrazol and uridine for culture to realize amplification of the adventitious buds, and (3) transferring stem segments obtained by proliferation culture into a rooting culture medium containing sodium nitroprusside and methyl jasmonate for culture to induce rooting. The efficient sterile rapid propagation system of the fenugreek established by the invention has the advantages that the differentiation rate of adventitious buds reaches 91.3%, the proliferation coefficient is 10.8, the rooting rate reaches 97.5%, the propagation speed is high, and high-quality seedlings can be produced in a large scale and the yield and quality are improved.

Inventors

  • Shi Yinji
  • PAERHATI.ROUZI
  • HE JIANG
  • CHENG HUAN
  • Sureyar Abdili Aizezi
  • MENG FANTAO
  • MA SHENGJUN

Assignees

  • 新疆维吾尔自治区药物研究院
  • 新疆农业大学

Dates

Publication Date
20260512
Application Date
20260316

Claims (10)

  1. 1. The method for establishing the efficient regeneration system of the fenugreek is characterized by comprising the following steps of: (1) Primary induction culture, namely inoculating the fenugreek explant into a primary induction culture medium for induction culture to generate adventitious buds, wherein the primary induction culture medium comprises a basic culture medium, a growth regulator, 10 -9 ~10 -11 M5-azacytidine, 0.1-0.5 mM spermidine and 3-8 mu M fluopicolide; (2) Proliferation culture, namely dividing adventitious buds, transferring the adventitious buds into a proliferation culture medium for culture to realize amplification of the adventitious buds, wherein the proliferation culture medium comprises a basic culture medium, a growth regulator, 1-3wt% of trehalose, 0.02-0.08 mg/L of paclobutrazol and 8-12 mu M of uridine; (3) Rooting culture, namely transferring the stem segments obtained by the proliferation culture in the step (2) into a rooting culture medium for culture, and inducing rooting, wherein the rooting culture medium comprises a basic culture medium, a growth regulator, 10-20 mu M sodium nitroprusside and 0.1-10 mu mol/L methyl jasmonate.
  2. 2. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 1, wherein the primary induction medium in the step (1) comprises a basal medium, a growth regulator, 10 -9 M5-azacytidine, 0.3 mM spermidine and 5 mu M fluazinone.
  3. 3. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 1 or 2, wherein the growth regulator in the primary induction medium in the step (1) comprises 0.5-1.5 mg/L of alpha-naphthylacetic acid and 0.1-0.8 mg/L of 6-benzylaminoadenine.
  4. 4. A method of establishing a high efficiency regeneration system for fenugreek according to claim 3, wherein the growth regulator in the primary induction medium in step (1) comprises 1.0 mg/L α -naphthylacetic acid and 0.5 mg/L6-benzylaminoadenine.
  5. 5. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 1, wherein the proliferation medium in the step (2) comprises basal medium, a growth regulator, 1 wt% of trehalose, 0.05 mg/L of paclobutrazol and 10 mu M of uridine.
  6. 6. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 1 or 5, wherein the growth regulator in the proliferation medium in the step (2) comprises 0.2-0.8 mg/L6-benzylaminoadenine and 0.1-0.5 mg/L alpha-naphthylacetic acid.
  7. 7. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 6, wherein the growth regulator in the proliferation medium in the step (2) comprises 0.6 mg/L of 6-benzylaminoadenine and 0.2. 0.2 mg/L of alpha-naphthylacetic acid.
  8. 8. The method for establishing the efficient regeneration system of fenugreek according to claim 1, wherein the rooting medium in the step (3) comprises a basal medium, a growth regulator, 15 mu M sodium nitroprusside and 5 mu mol/L methyl jasmonate.
  9. 9. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 1 or 8, wherein the growth regulator in the rooting medium in the step (3) comprises 0.5-1.5 mg/L of alpha-naphthylacetic acid and 0.2-0.8 mg/L of 6-benzylaminoadenine.
  10. 10. The method for establishing a high-efficiency regeneration system of fenugreek according to claim 9, wherein the growth regulator in the rooting medium in the step (3) comprises 1.0 mg/L of alpha-naphthylacetic acid and 0.5. 0.5 mg/L of 6-benzylaminoadenine.

Description

Method for establishing efficient regeneration system of fenugreek Technical Field The invention belongs to the technical field of medicinal plant tissue culture, and particularly relates to a method for establishing a high-efficiency regeneration system of fenugreek. Background Fenugreek (Trigonella foenum-graecum L.) is used as a core prescription medicinal material of the granules for treating asthma Zu Pa, and has special industrial value. The seed is rich in trigonelline, diosgenin, flavonoids, etc., has effects of lowering blood sugar, reducing blood lipid, reducing cholesterol, resisting oxidation, and relieving inflammation, and can be used for treating bronchitis, cough and nasal obstruction, cold hernia, abdominal distention, cold dampness tinea pedis, kidney essence deficiency, waist soreness, etc. The plant is about 60 cm a high, the root system is developed, the stem is upright, the feathered three leaves are formed, and the corolla is yellow white or light yellow. The pods are 7.5cm in average length and each pod contains 29 seeds, commonly known as bitter beans, fenugreek and the like. It is cultivated commercially in large areas in many countries. The fenugreek has drought resistance, cold resistance, heat resistance and the like. The plant tissue culture technology is an important basis for realizing in vitro rapid propagation, germplasm resource preservation and genetic transformation. At present, a plurality of researches and reports on tissue culture of fenugreek exist, but the prior art still has a plurality of defects, which are mainly characterized in that (1) the regeneration efficiency is low and the stability is poor, (2) the rooting and transplanting life rate is to be improved, and the like. Therefore, the development of a high-efficiency and stable fenugreek regeneration system is very significant for accelerating the breeding of excellent fenugreek germplasm. Disclosure of Invention In order to solve the defects in the prior art, the invention aims to provide a method for establishing a high-efficiency regeneration system of fenugreek, which can realize high-quality and high-efficiency breeding of fenugreek seedlings. The technical scheme for solving the technical problems is as follows, and the method for establishing the efficient regeneration system of the fenugreek comprises the following steps: (1) Primary induction culture, namely inoculating the fenugreek explant into a primary induction culture medium for induction culture to generate adventitious buds, wherein the primary induction culture medium comprises a basic culture medium, a growth regulator, 10 -9~10-11 M5-azacytidine, 0.1-0.5 mM spermidine and 3-8 mu M fluopicolide; (2) Proliferation culture, namely dividing adventitious buds, transferring the adventitious buds into a proliferation culture medium for culture to realize amplification of the adventitious buds, wherein the proliferation culture medium comprises a basic culture medium, a growth regulator, 1-3wt% of trehalose, 0.02-0.08 mg/L of paclobutrazol and 8-12 mu M of uridine; (3) Rooting culture, namely transferring the stem segments obtained by the proliferation culture in the step (2) into a rooting culture medium for culture, and inducing rooting, wherein the rooting culture medium comprises a basic culture medium, a growth regulator, 10-20 mu M sodium nitroprusside and 0.1-10 mu mol/L methyl jasmonate. The invention has the beneficial effects that the basic culture medium in the fenugreek regeneration system provides basic substances such as carbon sources, inorganic salts and the like, and specific active ingredients such as primary induction culture medium which mainly has the effect of inducing fenugreek explants to generate adventitious buds are respectively added in each culture stage, and the culture medium comprises the basic culture medium and a growth regulator, and 5-azacytidine, spermidine and fluazinone are also added. The 5-azacytidine captures DNA methyltransferase by incorporating into the DNA strand, resulting in DNA demethylation, making it easier for the cell to respond to signals from external growth regulators. In the primary induction stage, the explant is subjected to in vitro adversity stress, ABA (abscisic acid) is easy to accumulate endogenously, cell division and bud differentiation are inhibited, and the fluazinone reduces the ABA level in the explant by blocking the synthesis of the ABA, so that the inhibition effect on cell division and bud primordium is eliminated. Spermidine plays a role as a regulator of cell division and morphogenesis during tissue culture, and can stabilize the cell state. In the primary induction culture medium, 5-azacytidine is responsible for unlocking genes, fluazinone is responsible for eliminating inhibition signals, spermidine is responsible for stabilizing cell states and providing power for division, the direct induction effect of a growth regulator is combined, the four substances cooperate, a